The present invention relates to a novel protein having a kinase activity and a gene encoding said protein.
Antigen binding to the membrane IgR initiates the activation and maturation of the antigen-specific B cells in the peripheral lymphoid organs (Rajewsky, Nature (Lond.)., 381:751-758, 1996; Sakaguchi et al., Adv. Immunol. 54:337-392, 1993). B cells enter the outer periarterial lymphoid sheath (PALS) (Rajewsky, Nature (Lond.)., 381:751-758, 1996) and initiate costimulus-dependent interactions with specific Th cells and interdigitating dendritic cells within 48 h after immunization (MacLennan, Annu. Rev. Immunol. 12:117-139, 1994; Liu et al., Immunol. Rev. 156:111-126, 1997). Antigen-driven B cells proliferate in the outer PALS and then undergo further activation in the lymphoid follicles to establish the germinal center (herein sometimes abbreviated as GC) (Han et al., J. Immunol. 155:556-567, 1995; Jacob et al., J. Exp. Med. 176:679-687, 1992; Kelsoe, Immunity 4:107-111, 1996). Such B cells mature into large slgxe2x88x92 centroblasts that rapidly move through the cell cycle to form the dark zone and further mature into centrocytes that express a unique surface character of PNA+B220+slgM+slgDxe2x88x92CD38xe2x88x92 in the light zone of the GC (Kosco-Vilbois et al., 1997. Immunol. Today 18:225-230, 1997; Kelsoe, Immunol. Today 16:324-326, 1995; Oliver et al., J. Immunol. 158:1108-1115, 1997).
Centrocytes presumably undergo the processes of either apoptosis or affinity maturation of immunoglobulin V regions and the change process of class switching toward the IgG class antigen. Some centrocytes survive for a longer period in the lymphoid compartment as memory B cells. The other centrocytes probably migrate to the marginal zone of the GC and receive further antigenic stimulation and costimulatory signals through B cell activation molecules, such as CD40 and CD38, and receptors for various B cell stimulatory cytokines (Gray et al., J. Exp. Med., 180:141-155, 1994; Foy et al., J. Exp. Med., 180:157-163, 1994). Antigen-specific B cells further stimulated in this area probably migrate into the interstitial region of the spleen (called red pulp), where various kinds of other immune-competent cells may interact with antigen-driven B cells. Histochemical analysis in several autoimmune mice identified unique antibody-producing cells in this area which appear as plasma cells or aberrant plasma cells called Mott cells (Tarlinton et al., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J. Immunol., 158:992-997, 1997).
Autoimmunity is a phenomenon in which the impairment of self/nonself discrimination occurs frequently in the antigen-specific lymphocytes (Theofilopoulos, Immunol. Today, 16:90-98, 1995). The immune systems of various autoimmune diseases show the combinatory mechanism involving T cells and B cells (Theofilopoulos et al., Adv. Immunol., 37:269-290, 1985; Okamoto et al., J. Exp. Med. 175:71-79, 1992; Reininger et al., J. Exp. Med., 184:853-861, 1996; Theofilopoulos, et al., Immunol. Rev. 55:179-216, 1981; Watanabe-Fukunaga et al., Nature (Lond.)., 356:314-317, 1992; Takahashi et al., Cell, 76:969-976, 1994; Shlomchick et al., Nature (Lond.). 328:805-811, 1987).
NZB and NZW are the strains characterized by multiple genetic factors generating the severe autoimmune state of SLE as (NZBxc3x97NZW)F1 mice (Theofflopoulos et al., Adv. Immunol., 37:269-290, 1985; Okamoto et al., J. Exp. Med., 175:71-79, 1992; Reininger et al., J. Exp. Med., 184:853-861, 1996; Theofilopoulos et al., Immunol. Rev., 55:179-216, 1981). NZB mice spontaneously generate the state of autoimmunity with the anti-red blood cell antibody that causes an autoimmune hemolytic anemia (Okamoto et al., J. Exp. Med., 175:71-79, 1992). NZW mice show an insidious autoimmune phenomenon (Reininger et al., J. Exp. Med. 184:853-861, 1996). The SLE state of (NZBxc3x97NZW)F1 mice is apparently caused by multiple genetic factors associated with T and B cells (Theofilopoulos et al., Immunol. Rev., 55:179-216, 1981). NZB mice show an apparent abnormality of B cells, but the molecular mechanism of the abnormal B cell activation in NZB mice remains to be elucidated.
To address the issue of which molecules are involved in such maturation of B cells, the present inventors prepared monoclonal antibodies against intracellular components of a murine B cell line WEHI-231, which has the NZB genetic background. A monoclonal antibody named 29-15 recognizes a differentiation antigen whose expression is augmented in GC-B cells of peripheral lymphoid organs. With the 29-15 monoclonal antibody, the present inventors studied the expression of the antigen in peripheral lymphoid organs, which characterized the molecule as a differentiation antigen upregulated in the light zone of the GC from hyperimmunized mice. In the spleen of NZB mice, IgM-producing plasma cells with high expression of the GANP antigen appear before the onset of autoimmunity, which would suggest that this is an important molecular event for understanding the peripheral immune response and autoimmunity with autoantibodies.
The present inventors have studied to identify the above-mentioned antigen whose expression is selectively increased in centrocytes of germinal center, and confirmed by in situ RNA hybridization using an isolated cDNA probe (ganp probe) that the expression of ganp mRNA is increased in the area stained with 29-15 monoclonal antibody. It was also confirmed that the gene product, GANP protein, is a protein of 210 kD which is localized in cytoplasma and nucleus, and is structurally similar with a transcription regulating factor in yeasts, SAC3. When B cells are activated with anti-IgM antibody and anti-CD40 antibody, the amount of kinase which binds to GANP protein increased. These results suggests that GANP protein may be involved in a signal conversion of abnormal B cell differentiation in certain autoimmune state. The present invention has been completed on the basis of these findings.
Thus, the present invention provides a GANP protein represented by the amino acid sequence shown in SEQ ID No.1 or No.3 of the sequence listing. According to the present invention, there is provided a GANP mutant protein which is consisted of the amino acid sequence wherein one or more amino acids are deleted, one or more amino acids are substituted with other amino acid(s), and/or one or more other amino acids are added in the amino acid sequences shown in SEQ ID No.1 or No.3 of the sequence listing, and has a kinase activity similar with that of GANP protein. According to the present invention, there is provided a polypeptide which contains, as a partial sequence, a full length amino acid sequence of the aforementioned GANP protein or the aforementioned GANP mutant protein.
According to another aspect, the present invention provides a polynucleotide which encodes the aforementioned GANP protein or GANP mutant protein. The typical polynucleotide is DNA encoding GANP protein derived from mammal, and the DNA of mammal gene is preferred among them. Examples of most preferred polynucleotide are represented by the base sequences shown in SEQ ID No. 2 (DNA sequence encoding GANP protein from mouse) or SEQ ID No. 4 (DNA sequence encoding GANP protein from human) of the sequence listing.
Further, according to the present invention, there is provided an antisense polynucleotide which is composed of the base sequence of an antisense chain of the aforementioned polynucleotide, or derivatives of said antisense polynucleotide. Furthermore, according to the present invention, there is provided a polynucleotide or antisense polynucleotide of continuous 12 or more bases which is a partial sequence of the aforementioned polynucleotide or the aforementioned antisense polynucleotide, and a chemically modified polynucleotide or antisense polynucleotide of the aforementioned polynucleotide or the aforementioned antisense polynucleotide.
According to further another aspect, the present invention provides a method for obtaining DNA of the base sequence shown in SEQ ID No. 2 or No. 4 of the sequence listing or DNA which is the homologue from other mammal, wherein the aforementioned polynucleotide or antisense polynucleotide is used as a probe, and cDNA which hybridizes to the probe is obtained from mammal cDNA library. The length of the cDNA is almost the same as that of GANP gene, and the protein encoded by it has approximately 210 kDa. Further, according to the present invention, there is provided cDNA obtained by the aforementioned method and GANP protein encoded by it.
According to further another aspect of the present invention, there is provided an antibody which recognizes GANP protein or GANP mutant protein.